Fast chemical reactions may be initiated by rapidly mixing two reactants. The reactions may, in principle, be followed in one of two main ways. The first of these, called the continuous-flow method, employs standard detection techniques (as distinct from fast ones) but has the serious drawback of consuming relatively large amounts of reagents. Advances in electronics and the techniques for rapidly collecting data have caused the continuous-flow method to become less important than the stopped-flow technique, which uses relatively small amounts of the reagents.
In the stopped-flow apparatus, the reagents are stored in a pair of syringes which, to initiate the reaction, are driven forward by means of a hydraulic or pneumatic piston. The reactants then mix in a small mixing chamber before entering the reaction or observation cell where the course of the reaction is followed by an appropriate method. As the freshly mixed solution enters the reaction cell, the solution already there is forced into a third syringe (called the stopping syringe). The plunger of this syringe is thus driven onto a fixed stopping block which brings the flow of reagents to an abrupt stop. At this instant, a micro-switch is closed to trigger an electric impulse to start data collection.
All stopped-flow machines have these features in common. The various models usually differ only in the design and construction of the mixer, reaction cells and methods of detecting chemical changes.
The first commercially available stopped-flow apparatus, which is still the most popular one, was produced by the Durrum Instrument Company. It is based on a published design by Gibson and Milnes (Biochem. J. Vol. 91, 161 (1964)). Moderately satisfactory temperature control is achieved by circulating water through the metal block enclosing the reaction cell and by immersing the driving syringes in a shallow bath through which the same water is circulated. The apparatus has an excellent four or eight jet mixer close to the reaction cell, which has a long light path to enable studies with dilute solutions to be carried out. The flow system is mounted on a rigid metal casting to minimize effects due to vibrations set up when the flow is stopped. The standard apparatus has a set of adjustable mirrors which enable it to be used for both transmission and fluorescence studies. It is also available with a cell containing electrodes for temperature-jump work.
The main disadvantages of the apparatus are:
1. Temperature control is not good enough for many studies in physical and analytical chemistry. This deficiency could only be rectified by redesigning the apparatus. PA0 2. The reaction cells cannot be readily changed or modified to permit other means of detecting chemical changes. PA0 3. Corrosion of the valve stems and the plungers of the syringes is a frequent problem unless care is taken to dismantle and clean the apparatus regularly. PA0 1. Temperature control is hopelessly inadequate for precision work, despite the fact that thermostatted liquids may be circulated through the metal block in which the flow system is mounted. Part of the problem is that the plastic flow system and syringes are poor thermal conductors and the apparatus has no provision for preequilibrating the solutions. PA0 2. In use, the O-rings set in the Kel-F (trade mark) syringes soon become compressed and the apparatus develops leaks. This is not permissible in a stopped-flow apparatus. The problem becomes very pronounced when work is carried out at different temperatures, due to the different coefficients of expansion of the syringes and the O-rings. PA0 3. Provision is made in the apparatus for detecting light changes at right angles to the beam of light from the monochromator. However, many users of the apparatus have found that studies of light scattering or fluorescence are not possible owing to the fact that the Teflon (trade mark) parts of the reaction cell fluoresce very strongly. PA0 1. The whole flow system is made of glass, quartz, Teflon (trade mark) or Kel-F (trade mark). This ensures chemical inertness towards most materials. PA0 2. The storage reservoirs and the flow system are enclosed in a thermostatted bath whose temperature can be accurately adjusted, controlled and quickly changed as required. Temperature control to +0.005.degree. C. has been achieved in the range -10.degree. to +60.degree. C. PA0 3. The driving and stopping syringes, which are made of glass with Teflon (trade mark) luer-loc tips, are commercially available in different sizes. They may be easily interchanged to permit different volumes of the reagents to be mixed. PA0 4. The mixer is a specially designed unit to which reaction cells of different designs and materials can be quickly and firmly attached. In the mixer, double mixing of the reagents is achieved in about 1-2 milliseconds by dividing each stream of reagents before mixing them, at the entrance to a small mixing chamber, where the second mixing occurs. This chamber is situated at the entrance to the reaction cell. The overall dead-time when this mixer is attached to a reaction cell of 2 mm bore and 10 mm length is about 4 milliseconds when the driving cylinder is actuated by an air pressure of 20 lbs./sq. in. PA0 5. The pneumatic driving piston is of large bore and turbulent flow of the reactants is achieved at pressures between 15-20 lbs./sq.in. Such low pressures minimize problems due to shock waves, generated in most stopped-flow machines when the flow is abruptly halted. Shock waves are also further decreased in the present machine by the use of Teflon (trade mark) tubing between the syringes and the reaction chamber. PA0 6. A particularly important feature of the apparatus is that a number of different kinds of reaction cells are available. These can be easily and rapidly interchanged. This feature differs from conventional apparatus in that only the reaction cell and not the complete flow system is changed on order to modify the apparatus. At the present time, the following cells are available:
The American Instrument Company has introduced a stopped-flow apparatus with a number of attractive features such as small volume and easy attachment to different types of commercial spectrophotometers. Unfortunately, the apparatus has a number of undesirable weaknesses:
In 1974, Caldin, Crooks and Queen described a stopped-flow apparatus which, apart from the Teflon (trade mark) keys of the three-way stopcocks, was made entirely of glass or quartz. The syringes and stopcocks were mounted on a platform and connected by narrow bore glass tubes to the mixer and reaction cell which were suspended on mounts below the platform. The reaction chamber was connected to a source of monochromatic light and a photomultiplier by means of fibre-optic light guides (J. Phys. E., Scientific Instruments. 6, 930 (1973)). This arrangement allowed the mixer and reaction chamber to be immersed in a well-controlled constant temperature bath.
This apparatus is now commercially available from Nortech Laboratories U.K. In terms of temperature control and ease of use it is probably the most reliable apparatus available. The apparatus is constructed in modules. The driving piston and its controls, as well as the stopping block and triggering microswitch, are common to all machines. A number of different flow modules are available for light transmittance, fluorescence and temperature-jump studies. The apparatus is, therefore, quite versatile. However, each of the various modules is suitable for only one type of study and each one is quite expensive. The versatility of this apparatus is only available at considerable cost.